Compositions and methods for regulating angiogenesis

ABSTRACT

In accordance with the present invention, EC progenitors can be used in a method for regulating angiogenesis, i.e., enhancing or inhibiting blood vessel formation, in a selected patient and in some preferred embodiments for targetting specific locations. For example, the EC progenitors can be used to enhance angiogenesis or to deliver an angiogenesis modulator, e.g. anti- or pro-angiogenic agents, respectively to sites of pathologic or utilitarian angiogenesis. Additionally, in another embodiment, EC progenitors can be used to induce reendothelialization of an injured blood vessel, and thus reduce restenosis by indirectly inhibiting smooth muscle cell proliferation.

[0001] This application claims priority to U.S. patent application Ser.No. 09/228,020, filed Jan. 11, 1999 which claims priority to U.S.application Ser. No. 08/744,882, filed Nov. 8, 1996 now U.S. Pat. No.5,980,887.

FIELD OF THE INVENTION

[0002] The invention relates to methods for isolating and usingendothelial progenitor cells and compositions for use in the methods.

BACKGROUND OF THE INVENTION

[0003] Blood vessels are the means by which oxygen and nutrients aresupplied to living tissues and waste products removed from livingtissue. Angiogenesis is the process by which new blood vessels areformed, as reviewed, for example, by Folkman and Shing, J. Biol. Chem.267 (16), 10931-10934 (1992). Thus angiogenesis is a critical process.It is essential in reproduction, development and wound repair. However,inappropriate angiogenesis can have severe consequences. For example, itis only after many solid tumors are vascularized as a result ofangiogenesis that the tumors begin to grow rapidly and metastasize.Because angiogenesis is so critical to these functions, it must becarefully regulated in order to maintain health. The angiogenesisprocess is believed to begin with the degradation of the basementmembrane by proteases secreted from endothelial cells (EC) activated bymitogens such as vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (bFGF). The cells migrate and proliferate,leading to the formation of solid endothelial cell sprouts into thestromal space, then, vascular loops are formed and capillary tubesdevelop with formation of tight junctions and deposition of new basementmembrane.

[0004] In the adults, the proliferation rate of endothelial cells istypically low compared to other cell types in the body. The turnovertime of these cells can exceed one thousand days. Physiologicalexceptions in which angiogenesis results in rapid proliferation occursunder tight regulation are found in the female reproduction system andduring wound healing.

[0005] The rate of angiogenesis involves a change in the localequilibrium between positive and negative regulators of the growth ofmicrovessels. Abnormal angiogenesis occurs when the body loses itscontrol of angiogenesis, resulting in either excessive or insufficientblood vessel growth. For instance, conditions such as ulcers, strokes,and heart attacks may result from the absence of angiogenesis normallyrequired for natural healing. On the contrary, excessive blood vesselproliferation may favor tumor growth and spreading, blindness, psoriasisand rheumatoid arthritis.

[0006] The therapeutic implications of angiogenic growth factors werefirst described by Folkman and colleagues over two decades ago (Folkman,N. Engl. J. Med., 285:1182-1186 (1971)). Thus, there are instances wherea greater degree of angiogenesis is desirable—wound and ulcer healing.Recent investigations have established the feasibility of usingrecombinant angiogenic growth factors, such as fibroblast growth factor(FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) andBaffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial cell growthfactor (ECGF)(Pu, et al., J Surg Res, 54:575-83 (1993)), and morerecently, vascular endothelial growth factor (VEGF) to expedite and/oraugment collateral artery development in animal models of myocardial andhindlimb ischemia (Takeshita, et al., Circulation, 90:228-234 (1994) andTakeshita, et al., J Clin Invest, 93:662-70 (1994)).

[0007] Conversely, there are also instances, where inhibition ofangiogenesis is desirable. For example, many diseases are driven bypersistent unregulated angiogenesis. In arthritis, new capillary bloodvessels invade the joint and destroy cartilage. In diabetes, newcapillaries invade the vitreous, bleed, and cause blindness. Ocularneovascularization is the most common cause of blindness. Tumor growthand metastasis are angiogenesis-dependent. A tumor must continuouslystimulate the growth of new capillary blood vessels for the tumor itselfto grow.

[0008] The current treatment of these diseases is inadequate. Agentswhich prevent continued angiogenesis, e.g, drugs (TNP-470), monoclonalantibodies and antisense nucleic acids, are currently being tested.However, new agents that inhibit angiogenesis are needed.

[0009] Recently, the feasibility of gene therapy for modulatingangiogenesis has been demonstrated. For example, promoting angiogenesisin the treatment of ischemia was demonstrated in a rabbit model and inhuman clinical trials with VEGF using a Hydrogel-coated angioplastyballoon as the gene delivery system. Successful transfer and sustainedexpression of the VEGF gene in the vessel wall subsequently augmentedneovascularization in the ischemic limb (Takeshita, et al., LaboratoryInvestigation, 75:487-502 (1996); Isner, et al., Lancet, 348:370(1996)). In addition, it has been demonstrated that direct intramuscularinjection of DNA encoding VEGF into ischemic tissue inducesangiogenesis, providing the ischemic tissue with increased blood vessels(U.S. Ser. No. 08/545,998; Tsurumi et al., Circulation, In Press).

[0010] Alternative methods for regulating angiogenesis are stilldesirable for a number of reasons. For example, it is believed thatnative endothelial cell (EC) number and/or viability decreases overtime. Thus, in certain patient populations, e.g., the elderly, theresident population of ECs that is competent to respond to administeredangiogenic cytokines may be limited.

[0011] Moreover, while agents promoting or inhibiting angiogenesis maybe useful at one location, they may be undesirable at another location.Thus, means to more precisely regulate angiogenesis at a given locationare desirable.

SUMMARY OF THE INVENTION

[0012] We have now discovered that by using techniques similar to thoseemployed for HSCs, EC progenitors can be isolated from circulatingblood. In vitro, these cells differentiate into ECs. Indeed, one can usea multipotentiate undifferentiated cell as long as it is still capableof becoming an EC, if one adds appropriate agents to result in itdifferentiating into an EC.

[0013] We have also discovered that in vivo, heterologous, homologous,and autologous EC progenitor grafts incorporate into sites of activeangiogenesis or blood vessel injury, i.e. they selectively migrate tosuch locations. This observation was surprising. Accordingly, one cantarget such sites by the present invention.

[0014] The present invention provides a method for regulatingangiogenesis in a selected patient in need of a change in the rate ofangiogenesis at a selected site. The change in angiogenesis necessarymay be reduction or enhancement of angiogenesis. This is determined bythe disorder to be treated. In accordance with the method of the presentinvention, an effective amount of an endothelial progenitor cell ormodified version thereof to accomplish the desired result isadministered to the patient.

[0015] In order to reduce undesired angiogenesis, for example, in thetreatment of diseases such as rheumatoid arthritis, psoriasis, ocularneovascularization, diabetic retinopathy, neovascular glaucoma,angiogenesis-dependent tumors and tumor metastasis, a modifiedendothelial cell, having been modified to contain a compound thatinhibits angiogenesis, e.g., a cytotoxic compound or angiogenesisinhibitor, can be administered.

[0016] To enhance angiogenesis, for example in the treatment ofcerebrovascular ischemia, renal ischemia, pulmonary ischemia, limbischemia, ischemic cardiomyopathy and myocardial ischemia, endothelialprogenitor cells are administered. To further enhance angiogenesis anendothelial progenitor cell modified to express an endothelial cellmitogen may be used. Additionally, an endothelial cell mitogen or anucleic acid encoding an endothelial cell mitogen can further beadministered.

[0017] In another embodiment, the present invention provides methods ofenhancing angiogenesis or treating an injured blood vessel. Inaccordance with these methods, endothelial progenitor cells are isolatedfrom the patient, preferably from peripheral blood, and readministeredto the patient. The patient may also be treated with endothelial cellmitogens to enhance endothelial cell growth. The vessel injury can bethe result of balloon angioplasty, deployment of an endovascular stentor a vascular graft.

[0018] The present invention also provides a method of screening for thepresence of ischemic tissue or vascular injury in a patient. The methodinvolves contacting the patient with a labelled EC progenitor anddetecting the labelled cells at the site of the ischemic tissue orvascular injury.

[0019] The present invention also includes pharmaceutical products andkit for all the uses contemplated in the methods described herein.

[0020] Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGS. 1A-1G show cell shape and formation. FIG. 1A shows spindleshaped CD34+ attaching cells (AT^(CD34+)) 7 days after plating CD34+mononuclear blood cells (MB^(CD34+)) on fibronectin with standardmedium. Network formation (FIG. 1B) and cord-like structures (FIG. 1C)were observed 48 hours after plating co-culture of MB^(CD34+), labeledwith DiI dye (Molecular Probe), and unlabeled MB^(CD34−) (ratio of1:100) on fibronectin-coated dish. These cords consisted principally ofDiI-labeled MB^(CD) ³⁴⁺ derived cells (AT^(CD34+)). Beginning at 12hours after co-culture, MB^(CD34+) derived cells demonstrated multiplefoci of cluster formation (FIGS. 1D, 1E). AT^(CD34+) sprout from theperiphery, while round cells remain in the center and detach from thecluster several days later. After 5 days, uptake of labeled acetylatedlow density lipoprotein, acLDL-DiI (Molecular Probe). was seen inAT^(CD34+) cells at the periphery but not at the center of the cluster(FIGS. 1F, 1G).

[0022]FIG. 2 shows the number of AT^(CD34+) 12 hours and 3 days aftersingle culture of MB^(CD34+) on plastic alone (CD34±non), collagencoating (CD34⁺/COL), or fibronectin (CD34⁺/FN), and MB^(CD34−) onfibronectin (CD34⁻/FN). AT^(CD34+) yielded significantly higher numberof cells at 12 hours and 3 days when plated on fibronectin (p<0.05, byANOVA).

[0023]FIG. 3 shows FACS analysis of freshly isolated MB^(CD34+),AT^(CD34+) after 7 days in culture, and HUVECs. Cells were labeled withFITC using antibodies against CD34, CD31 (Biodesign); Flk-1, Tie-2(Santa Cruz Biotechnology); and CD45. All results were confirmed bytriplicate experiments. Shaded area of each box denotes negative antigengate; white area denotes positive gate. Numbers indicated for individualgates denote percentage of cells determined by comparison withcorresponding negative control labeling.

[0024]FIG. 4 shows expression of ecNOS mRNA in MB^(CD34−), MB^(CD34+),AT^(CD34+), human coronary smooth muscle cells (HCSMCs) and HUVECs. DNAwas reverse transcribed from ≈1×10⁶ cells each. Equal aliquots of theresulting DNA were amplified by PCR (40 cycles) with paired primers(sense/antisense: AAG ACA TTT TCG GGC TCA CGC TGC GCA CCC/TGG GGT AGGCAC TTT AGT AGT TCT CCT AAC, SEQ ID NO: 1) to detect ecNOS mRNA. Equalaliquots of the amplified product were analyzed on a 1% agarose gel.Only a single band was observed, corresponding to the expected size (548bp) for ecNOS. Lane 1=MB^(CD34−); Lane 2=MB^(CD34+); Lane 3=AT^(CD34+)after 3d; Lane 4=AT^(CD34+) after 7d; Lane 5=HCSMCs; Lane 6=HUVECs.

[0025]FIG. 5 is a graph illustrating NO release from AT^(CD34+) wasmeasured with an NO-specific polarographic electrode connected to an NOmeter (Iso-NO, World Precision Instruments) (Shibuki and Okada, Nature358, 676 (1991).). Calibration of NO electrode was performed dailybefore experimental protocol according to the following equation:2KNO₂+2KI+2H₂SO₄→2NO+I₂+2H₂O+2K₂SO₄. A standard calibration curve wasobtained by adding graded concentrations of KNO₂(0-500 nmol/L) tocalibration solution containing KI and H₂SO4. Specificity of theelectrode to NO was previously documented by measurement of NO fromauthentic NO gas (Weyrich, et al, Circ. Res. 75, 692 (1994)). AT^(CD34+)cultured in 6-well plate were washed and then bathed in 5 ml of filteredKrebs-Henseleit solution. Cell plates were kept on a slide warmer (LabLine Instruments) to maintain temperature between 35 and 37° C. For NOmeasurement, sensor probe was inserted vertically into the wells, andthe tip of the electrode remained 2 mm under the surface of thesolution. Measurement of NO, expressed as pmol/10⁵ cells, was performedin a well with incremental doses of VEGF (1, 10, 100 ng/ml) and Ach(0.1, 1, 10 μM). HUVECs and bovine aortic ECs were employed as positivecontrols. For negative control, HCSMCs, NO was not detectable. Allvalues reported represent means of 10 measurements for each group.

[0026] FIGS. 6A-6D show co-culture of MB^(CD34+) with HUVECs. Freshlyisolated MB^(CD34+) were labeled with DiI dye and plated on a confluentHUVEC monolayer attached to a fibronectin-coated chamber slide at adensity of 278 cells/mm² (Nunc). Differentiation of MB^(CD34+) intospindle shaped attaching cells (AT^(CD34+)) (red fluorescence) wasobserved among HUVECs within 12 h (FIG. 6A). The AT^(CD34+) numberincreased on monolayer for 3 days (FIG. 6B), while meshwork structureswere observed in some areas (FIG. 6C). Three days after co-culture, bothcells were re-seeded on Matrigel (Becton Dickinson)-coated slides andwithin 12 h disclosed capillary network formation, consisting ofDiI-labeled AT^(CD34+) and HUVECs (FIG. 6D).

[0027]FIG. 7 shows the effect of activated ECs and VEGF on MB^(CD34+)differentiation was investigated by pretreatment of HUVEC with TNF-α (20ng/ml) for 12 hours, and/or incubation of AT^(CD34+)/HUVEC co-culturewith VEGF (50 ng/ml).

[0028] FIGS. 8A-8K show sections retrieved from ischemic hindlimbfollowing in vivo administration of heterologous (FIGS. 8A-8H),homologous (FIG. 8I), and autologous (FIGS. 8J, 8K) EC progenitors.FIGS. 8A, 8B: Red fluorescence in small inter-muscular artery 6 wksafter injection of DiI-labeled MB^(CD34+). Green fluorescence denotesEC-specific lectin UEA-1. FIG. 8C: DiI (red) and CD31 (green) incapillaries between muscles, photographed through double filter 4 wksafter DiI-labeled MB^(CD34+) injection. FIG. 8D: Same capillarystructure as in FIG. 8C, showing CD31 expression by MB^(CD34+) whichhave been incorporated into host capillary structures expressing CD31.FIGS. 8E, 8F: Immunostaining 2 wks after MB^(CD34+) injection showscapillaries comprised of DiI-labeled MB^(CD34+) derived cells expressingtie-2 receptor (green fluorescence). Most MB^(CD34+) derived cells aretie-2 positive, and are integrated with some tie-2 positive native(host) capillary cells identified by absence of red fluorescence. FIGS.8G, 8H: Two wks after injection of DiI-labeled MB^(CD34−). Althoughisolated MB^(CD34−) derived cells (red) can be observed between muscles,but these cells do not express CD31. FIG. 8I: Immunohistochemicalβ-galactosidase staining of muscle harvested from ischemic limb ofB6,129 mice 4 wks following administration of MB^(Flk-1+) isolated fromβ-galactosidase transgenic mice. Cells overexpressing β-galactosidase(arrows) have been incorporated into capillaries and small arteries;these cells were identified as ECs by anti-CD31 antibody and BS-1lectin. FIGS. 8J,8K: Sections of muscles harvested from rabbit ischemichindlimb 4 wks after administration of autologous MB^(CD34+). DiIfluorescence (FIG. 8J) indicates localization of MB^(CD34+) derivedcells in capillaries seen in phase contrast photomicrograph (FIG. 8K).Each scale bar indicates 50 μm.

[0029]FIG. 9 is a photograph from a scanning electron microscope showingthat EC progenitors had adhered to the denuded arterial surface andassumed a morphology suggestive of endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

[0030] We have now discovered a means to regulate angiogenesis, topromote angiogenesis in certain subject populations, and to moreprecisely target certain tissues. These methods all involve the use ofendothelial cell progenitors. One preferred progenitor cell is anangioblast.

[0031] Post-natal neovascularization is believed to result exclusivelyfrom the proliferation, migration, and remodeling of fullydifferentiated endothelial cells (ECs) derived from pre-existing nativeblood vessels (Folkman, et al., Circ. Res. 28, 671 (1971); W. Risau,FASEB J. 9, 926 (1995)). This adult paradigm, referred to asangiogenesis, contrasts with vasculogenesis, the term applied toformation of embryonic blood vessels from EC progenitors (Risau, et al.,Development 105, 473 (1989).).

[0032] In contrast to angiogenesis, vasculogenesis typically begins as acluster formation, or blood island, comprised of EC progenitors (e.g.angioblasts) at the periphery and hematopoietic stem cells (HSCs) at thecenter (Flamme and Risau, Development 116, 435 (1992)). In addition tothis intimate and predictable spatial association, such EC progenitorsand HSCs share certain common antigenic determinants, including flk-1,tie-2, and CD-34. Consequently, these progenitor cells have beeninterpreted to derive from a common hypothetical precursor, thehemangioblast (Flamme and Risau, 1992, supra; His, et al, J. Clin.Invest. 97, 591 (1996)).

[0033] The demonstration that transplants of HSCs derived fromperipheral blood can provide sustained hematopoietic recoveryconstitutes inferential evidence for circulating stem cells. (Brugger,et al., J. Clin. Oncol. 12, 28 (1994)). This observation is now beingexploited clinically as an alternative to bone marrow transplantation.

[0034] We have now discovered that by using techniques similar to thoseemployed for HSCs, EC progenitors can be isolated from circulatingblood. In vitro, these cells differentiate into ECs. Indeed, one can usea multipotentiate undifferentiated cell as long as it is still capableof becoming an EC, if one adds appropriate agents to result in itdifferentiating into an EC.

[0035] We have also discovered that in vivo, heterologous, homologous,and autologous EC progenitor grafts incorporate into sites of activeangiogenesis or blood vessel injury, i.e., they selectively migrate tosuch locations. This observation was surprising. Accordingly, one cantarget such sites by the present invention.

[0036] In accordance with the present invention, EC progenitors can beused in a method for regulating angiogenesis, i.e., enhancing orinhibiting blood vessel formation, in a selected patient and in somepreferred embodiments for targetting specific locations. For example,the EC progenitors can be used to enhance angiogenesis or to deliver anangiogenesis modulator, e.g. anti- or pro-angiogenic agents,respectively to sites of pathologic or utilitarian angiogenesis.Additionally, in another embodiment, EC progenitors can be used toinduce reendothelialization of an injured blood vessel, and thus reducerestenosis by indirectly inhibiting smooth muscle cell proliferation. Ina still another embodiment, EC progenitors can be used to regeneratetissue, such as cardiac tissue.

[0037] In one preferred embodiment the EC cells can be used alone topotentiate angiogeneis in a patient. Some patient populations, typicallyelderly patients, may have either a limited number of ECs or a limitednumber of functional ECs. Thus, if one desires to promote angiogenesis,for example, to stimulate vascularization by using a potent angiogenesispromotor such as VEGF, such vascularization can be limited by the lackof ECs. However, by administering the EC progenitors one can potentiatevascularization in those patients.

[0038] Accordingly, the present method permits a wide range ofstrategies designed to modulate angiogenesis such as promotingneovascularization of ischemic tissues (Isner, et al, Lancet 348, 370(1996)). EC mitogens such as VEGF and bFGF, for example, have beenemployed to stimulate native ECs to proliferate, migrate, remodel andthereby form new sprouts from parent vessels (D′Amore and Thompson,Annu. Rev. Physiol. 49, 453 (1987)). A potentially limiting factor insuch therapeutic paradigms is the resident population of ECs that iscompetent to respond to administered angiogenic cytokines. The findingthat NO production declines as a function of age (Tschudi, et al, J.Clin. Invest. 98, 899 (1996)) may indicate a reduction in EC numberand/or viability that could be addressed by autologous EC grafting. Thesuccess demonstrated to date with autologous grafts of HSCs derived fromperipheral blood (Brugger, et al., 1995, supra) Kessinger and Armitage,Blood 77, 211 (1991); Sheridan, et al., Lancet 339, 640 (1992); Shpall,et al., J. Clin. Oncol. 12, 28 (1994)) supports the clinical feasibilityof a “supply side” approach to therapeutic angiogenesis. The in vivodata set forth herein indicate that autologous EC transplants arefeasible, and the in vitro experiments indicate that EC progenitors(MB^(CD34+)-derived ECs) can be easily manipulated and expanded ex vivo.

[0039] Our discovery that these EC progenitors home to foci ofangiogenesis makes these cells useful as autologous vectors for genetherapy and diagnosis of ischemia or vacular injury. For example, thesecells can be utilized to inhibit as well as augment angiogenesis. Foranti-neoplastic therapies, for example, EC progenitors can betransfected with or coupled to cytotoxic agents, cytokines orco-stimulatory molecules to stimulate an immune reaction, otheranti-tumor drugs or angiogenesis inhibitors. For treatment of regionalischemia, angiogenesis could be amplified by prior transfection of ECprogenitors to achieve constitutive expression of angiogenic cytokinesand/or selected matrix proteins (Sato, et al, Exp. Cell Res. 204, 223(1993); Pepper, et al., Biochem Biophys Res Comm 181, 902 (1991);Senger, et al, Am. J. Pathol. 149, 293 (1996)). In addition, the ECprogenitors may be labelled, e.g., radiolabelled, administered to apatient and used in the detection of ischemic tissue or vacular injury.

[0040] EC progenitors may be obtained from human mononuclear cellsobtained from peripheral blood or bone marrow of the patient beforetreatment. EC progenitors may also be obtained from heterologous orautologous umbilical cord blood. Peripheral blood is preferred due toconvenience. The leukocyte fraction of peripheral blood is mostpreferred. EC progenitors may be isolated using antibodies thatrecognize EC progenitor specific antigens on immature humanhematopoietic progenitor cells (HSCs). For example, CD34 is commonlyshared by EC progenitor and HSCs. CD34 is expressed by all HSCs but islost by hematopoietic cells as they differentiate (Civin, et al, JImmunol 133, 157 (1984); Katz, et al., Leuk. Res. 9, 191 (1985);Andrews, et al., Blood 67, 842 (1986)). It is also expressed by many,including most activated, ECs in the adult (Fina, et al, Blood 75, 17(1990); Soligo, et al, Leukemia 5, 1026 (1991); Ito, et al., Lab.Invest. 72, 532 (1995)). Flk-1, a receptor for vascular endothelialgrowth factor (VEGF) (deVries, et al, Science 255, 989 (1992); Terman,et al, Biochem. Biophys. Res. Commun. 187, 1579 (1992); Shalaby, et al,Nature 376, 62 (1995)), is also expressed by both early HSCs and ECs,but ceases to be expressed in the course of hematopoieticdifferentiation (Matthews, et al, Proc. Natl. Acad. Sci. USA. 88, 9026(1991); Millauer, et al, Cell 72, 835 (1993); Yamaguchi, et al.,Development 118, 489 (1993)).

[0041] To obtain the EC progenitors from peripheral blood about 5 ml toabout 500 ml of blood is taken from the patient. Ppreferably, about 50ml to about 200 ml of blood is taken.

[0042] EC progenitors can be expanded in vivo by administration ofrecruitment growth factors, e.g., GM-CSF and IL-3, to the patient priorto removing the progenitor cells.

[0043] Methods for obtaining and using hematopoietic progenitor cells inautologous transplantation are disclosed in U.S. Pat. No. 5,199,942, thedisclosure of which is incorporated by reference.

[0044] Once the progenitor cells are obtained by a particular separationtechnique, they may be administered to a selected patient to treat anumber of conditions including, but not limited to: unregulatedangiogenesis, blood vessel injury, ischemia, myocardial infarction,myocardial injury, restoration of left ventricular (LV) function,vascular occlusion or stenosis, peripheral vascular disease, sickle cellanemia, thalassemia, and the various conditions discussued suprarequiring inducing or enhancing angiogenesis. Additionally, theprogenitor cells may be used to vascularize grafts of transplantedtissue (e.g., such as skin grafts), to regenerate cells, to formbioprostheses (see, e.g., as discussed in U.S. Pat. No. 6,375,680), andin surgical procedures such as CABG. The progenitor cells may also beused to deliver inhibitory agents to tissues and cells in need, todecrease angiogeneis where desirable, e.g., to treat conditions such astumor growth, diabetic retinopathy, rheumatoid arthritis, and chronicinflammatory diseases (see, U.S. Pat. No. 5,318,957; Yancopoulos, et al.Cell 93: 661-4 (1998); Folkman, et al., Cell 87: 1153-5 (1996); andHanahan, et al., Cell 86: 353-64 (1996)).

[0045] The cells may be stored under cryogenic conditions. Optionally,the cells may be expanded ex vivo using, for example, the methoddisclosed by U.S. Pat. No. 5,541,103, the disclosure of which isincorporated by reference.

[0046] Preferably, the progenitor cells obtained from the patient arereadministered. Generally, from about 10⁶ to about 10¹⁸ progenitor cellsare administered to the patient.

[0047] The progenitor cells are administered to the patient by anysuitable means, including, for example, intravenous infusion, bolusinjection, and site-directed delivery via a catheter (e.g., such as anendocardial delivery catheter), stent, syringe, transthoracic drugdelivery device (U.S. Pat. No. 6,517,527) or other medical access deviceor implantable device. As discussed above, progenitor cells may beadministered with mitogens (e.g., such as VEGF), other agents formodulating angiogenesis (e.g., proteins, peptides, nucleic acids, drugs,small molecules and the like), and even other types of progenitor cells(see, e.g., U.S. patent Publication Ser. No. 20,020,142,457). Mitogensand agents may be delivered through the lumen of a medical access deviceand/or may coat at least a portion of the inner and/or outer walls ofsuch a device.

[0048] In certain aspects, it is desirable to deliver an endothelialprogenitor to specific cells in need of angiogenesis within a tissue. Inone aspect, endothelial progenitor cells are delivered to zones ofhibernating myocardium or to sites of myocardial infarction to preserveLV function.

[0049] For example, cells in need of angiogenesis may be identified byendomyocardial or electromechanical mapping (EMM) of cardiac tissueusing, for example, the NOGA system (Biosense-Webster) of catheter-basedmapping and navigation (see, Kawamoto, et al., Circulation 107(3), 461-8(2003), and references cited therein). The editing of the raw data maybe performed by a suitable system such as the NOGA system computer.

[0050] After mapping, a suitable medical access device capable ofdelivering one or more endothelial progenitor cells (a “deliverydevice”) (e.g., such as a percutaneous injection catheter) is positionedin proximity to cell(s) in need (such as at a zone of ischemia)identified by mapping. An intracardiac electrogram may be obtained todetect transient myocardial injury and/or premature ventricularcontractions as evidence of penetration of at least a portion of thedelivery device into the myocardium. After positioning, progenitor cellsare delivered (e.g., by injection) and the process can be repeated againas necessary to deliver additional progenitors to additional cell(s) inneed. ECG monitoring and creatine kinase monitoring and other assays maybe used to monitor the general cardiac health of the patient. SPECTMyocardial Perfusion studies may be performed, as known in the art, tomonitor angiogenesis.

[0051] Depending on the use of the progenitor cells, various geneticmaterial may be delivered to the cell. The genetic material that isdelivered to the EC progenitors may be genes, for example, those thatencode a variety of proteins including anticancer agents. Such genesinclude those encoding various hormones, growth factors, enzymes,cytokines, receptors, MHC molecules, telomerase, and the like. The term“genes” includes nucleic acid sequences both exogenous and endogenous tocells into which a virus vector, for example, a pox virus such as swinepox containing the human TNF gene may be introduced.

[0052] Additionally, it is of interest to use genes encodingpolypeptides for secretion from the EC progenitors so as to provide fora systemic effect by the protein encoded by the gene. Specific genes ofinterest include those encoding TNF, TGF-α, TGF-β, hemoglobin,interleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-11, interleukin-12 etc.,GM-CSF, G-CSF, M-CSF, human growth factor, co-stimulatory factor B7,insulin, factor VIII, factor IX, PDGF, EGF, NGF, IL-ira, EPO, β-globin,EC mitogens and the like, as well as biologically active muteins ofthese proteins. The gene may further encode a product that regulatesexpression of another gene product or blocks one or more steps in abiological pathway. In addition, the gene may encode a toxin fused to apolypeptide, e.g., a receptor ligand, or an antibody that directs thetoxin to a target, such as a tumor cell. Similarly, the gene may encodea therapeutic protein fused to a targeting polypeptide, to deliver atherapeutic effect to a diseased tissue or organ.

[0053] The cells can also be used to deliver genes to enhance theability of the immune system to fight a particular disease or tumor. Forexample, the cells can be used to deliver one or more cytokines (e.g.,IL-2) to boost the immune system and/or one or more antigens.

[0054] These cells may also be used to selectively administer drugs,such as an antiangiogenesis compound such as O-chloroacetyl carbamoylfumagillol (TNP-470). Preferably the drug would be incorporated into thecell in a vehicle such as a liposome, a timed released capsule, etc. TheEC progenitor would then selectively hone in on a site of activeangiogenesis such as a rapidly growing tumor where the compound would bereleased. By this method, one can reduce undesired side effects at otherlocations.

[0055] In one embodiment, the present invention may be used to enhanceblood vessel formation in ischemic tissue, i.e., a tissue having adeficiency in blood as the result of an ischemic disease. Such tissuescan include, for example, muscle, brain, kidney and lung. Ischemicdiseases include, for example, cerebrovascular ischemia, renal ischemia,pulmonary ischemia, limb ischemia, ischemic cardiomyopathy andmyocardial ischemia.

[0056] If it is desirable to further enhance angiogenesis, endothelialcell mitogens may also be administered to the patient in conjunctionwith, or subsequent to, the administration of the EC progenitor cells.Endothelial cell mitogens can be administered directly, e.g.,intra-arterially, intramuscularly, or intravenously, or nucleic acidencoding the mitogen may be used. See, Baffour, et al., supra (bFGF);Pu, et al, Circulation, 88:208-215 (1993) (aFGF); Yanagisawa-Miwa, etal., supra (bFGF); Ferrara, et al., Biochem. Biophys. Res. Commun.,161:851-855 (1989) (VEGF); (Takeshita, et al., Circulation, 90:228-234(1994)).

[0057] The nucleic acid encoding the EC mitogen can be administered to ablood vessel perfusing the ischemic tissue or to a site of vascularinjury via a catheter, for example, a hydrogel catheter, as described byU.S. Ser. No. 08/675,523, the disclosure of which is herein incorporatedby reference.

[0058] The nucleic acid also can be delivered by injection directly intothe ischemic tissue using the method described in U.S. Ser. No.08/545,998.

[0059] As used herein the term “endothelial cell mitogen” means anyprotein, polypeptide, mutein or portion that is capable of, directly orindirectly, inducing endothelial cell growth. Such proteins include, forexample, acidic and basic fibroblast growth factors (aFGF and bFGF),vascular endothelial growth factor (VEGF), epidermal growth factor(EGF), transforming growth factor α and β (TGF-α and TFG-β),platelet-derived endothelial growth factor (PD-ECGF), platelet-derivedgrowth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growthfactor (HGF), insulin like growth factor (IGF), erythropoietin, colonystimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophageCSF (GM-CSF) and nitric oxidesynthase (NOS). See, Klagsbrun, et al.,Annu. Rev. Physiol., 53, 217-239 (1991); Folkmnan, et al., J. Biol.Chem., 267, 10931-10934 (1992) and Symes, et al., Current Opinion inLipidology, 5, 305-312 (1994). Muteins or fragments of a mitogen may beused as long as they induce or promote EC cell growth.

[0060] Preferably, the endothelial cell mitogen contains a secretorysignal sequence that facilitates secretion of the protein. Proteinshaving native signal sequences, e.g., VEGF, are preferred. Proteins thatdo not have native signal sequences, e.g., bFGF, can be modified tocontain such sequences using routine genetic manipulation techniques.See, Nabel et al., Nature, 362, 844 (1993).

[0061] The nucleotide sequence of numerous endothelial cell mitogens,are readily available through a number of computer databases, forexample, GenBank, EMBL and Swiss-Prot. Using this information, a DNAsegment encoding the desired may be chemically synthesized or,alternatively, such a DNA segment may be obtained using routineprocedures in the art, e.g., PCR amplification. A DNA encoding VEGF isdisclosed in U.S. Pat. No. 5,332,671, the disclosure of which is hereinincorporated by reference.

[0062] In certain situations, it may be desirable to use nucleic acidsencoding two or more different proteins in order optimize thetherapeutic outcome. For example, DNA encoding two proteins, e.g., VEGFand bFGF, can be used, and provides an improvement over the use of bFGFalone. Or an angiogenic factor can be combined with other genes or theirencoded gene products to enhance the activity of targeted cells, whilesimultaneously inducing angiogenesis, including, for example, nitricoxide synthase, L-arginine, fibronectin, urokinase, plasminogenactivator and heparin.

[0063] The term “effective amount” means a sufficient amount ofcompound, e.g. nucleic acid delivered to produce an adequate level ofthe endothelial cell mitogen, i.e., levels capable of inducingendothelial cell growth and/or inducing angiogenesis. Thus, theimportant aspect is the level of mitogen expressed. Accordingly, one canuse multiple transcripts or one can have the gene under the control of apromoter that will result in high levels of expression. In analternative embodiment, the gene would be under the control of a factorthat results in extremely high levels of expression, e.g., tat and thecorresponding tar element.

[0064] The EC progenitors may also be modified ex vivo such that thecells inhibit angiogenesis. This can be accomplished, for example, byintroducing DNA encoding angiogenesis inhibiting agents to the cells,using for example the gene transfer techniques mentioned herein.Angiogenesis inhibiting agents include, for example, proteins such asthrombospondin (Dameron, et al., Science 265, 1582-1584 (1994)),angiostatin (O'Reilly, et al., Cell 79,315-328 (1994), IFN-alpha(Folkman, J. Nature Med. 1:27-31 (1995)), transforming growth factorbeta, tumor necrosis factor alpha, human platelet factor 4 (PF4);substances which suppress cell migration, such as proteinase inhibitorswhich inhibit proteases which may be necessary for penetration of thebasement membrane, in particular, tissue inhibitors of metalloproteinaseTIMP-1 and TIMP-2; and other proteins such as protamine which hasdemonstrated angiostatic properties, decoy receptors, drugs such asanalogues of the angioinhibin fumagillin, e.g., TNP-470 (Ingber, et al.,Nature 348, 555-557 (1990), antibodies or antisense nucleic acid againstangiogenic cytokines such as VEGF. Alternatively, the cells may becoupled to such angiogenesis inhibiting agent.

[0065] If the angiogenesis is associated with neoplastic growth the ECprogenitor cell may also be transfected with nucleic acid encoding, orcoupled to, anti-tumor agents or agents that enhance the immune system.Such agents include, for example, TNF, cytokines such as interleukin(IL) (e.g., IL-2, IL-4, IL-10, IL-12), interferons (IFN) (e.g., IFN-γ)and co-stimulatory factor (e.g., B7). Preferably, one would use amultivalent vector to deliver, for example, both TNF and IL-2simultaneously.

[0066] The nucleic acids are introduced into the EC progenitor by anymethod which will result in the uptake and expression of the nucleicacid by the cells. These can include vectors, liposomes, naked DNA,adjuvant-assisted DNA, catheters, gene gun, etc. Vectors includechemical conjugates such as described in WO 93/04701, which hastargeting moiety (e.g. a ligand to a cellular surface receptor), and anucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNAor RNA viral vector), fusion proteins such as described in WO 95/22618,which is a fusion protein containing a target moiety (e.g. an antibodyspecific for a target cell) and a nucleic acid binding moiety (e.g. aprotamine), plasmids, phage, etc. The vectors can be chromosomal,non-chromosomal or synthetic.

[0067] Preferred vectors include viral vectors, fusion proteins andchemical conjugates. Retroviral vectors include moloney murine leukemiaviruses and HIV-based viruses. One preferred HIV-based viral vectorcomprises at least two vectors wherein the gag and pol genes are from anHIV genome and the env gene is from another virus. DNA viral vectors arepreferred. These vectors include pox vectors such as orthopox or avipoxvectors, herpesvirus vectors such as a herpes simplex I virus (HSV)vector [Geller, et al., J. Neurochem, 64, 487 (1995); Lim, F., et al.,in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press,Oxford England) (1995); Geller, et al., Proc Natl. Acad. Sci.: USA.90,7603 (1993); Geller, et al., Proc Natl. Acad. Sci USA: 87, 1149 (1990)],Adenovirus Vectors [LaSalle, et al., Science, 259, 988 (1993); Davidson,et al., Nat. Genet 3, 219 (1993); Yang, et al., J. Virol. 69, 2004(1995)] and Adeno-associated Virus Vectors [Kaplitt, M. G., et al., Nat.Genet. 8, 148 (1994)].

[0068] Pox viral vectors introduce the gene into the cells cytoplasm.Avipox virus vectors result in only a short term expression of thenucleic acid. Adenovirus vectors, adeno-associated virus vectors andherpes simplex virus (HSV) vectors are preferred for introducing thenucleic acid into neural cells. The adenovirus vector results in ashorter term expression (about 2 months) than adeno-associated virus(about 4 months), which in turn is shorter than HSV vectors. Theparticular vector chosen will depend upon the target cell and thecondition being treated. The introduction can be by standard techniques,e.g., infection, transfection, transduction or transformation. Examplesof modes of gene transfer include e.g., naked DNA, CaPO₄ precipitation,DEAE dextran, electroporation, protoplast fusion, lipofecton, cellmicroinjection, viral vectors and use of the “gene gun”.

[0069] To simplify the manipulation and handling of the nucleic acidencoding the protein, the nucleic acid is preferably inserted into acassette where it is operably linked to a promoter. The promoter must becapable of driving expression of the protein in cells of the desiredtarget tissue. The selection of appropriate promoters can readily beaccomplished. Preferably, one would use a high expression promoter. Anexample of a suitable promoter is the 763-base-pair cytomegalovirus(CMV) promoter. The Rous sarcoma virus (RSV) (Davis, et al., Hum. GeneTher. 4, 151 (1993)) and MMT promoters may also be used. Certainproteins can expressed using their native promoter. Other elements thatcan enhance expression can also be included such as an enhancer or asystem that results in high levels of expression such as a tat gene andtar element. This cassette can then be inserted into a vector, e.g., aplasmid vector such as pUC118, pBR322, or other known plasmid vectors,that includes, for example, an E. coli origin of replication. See,Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, (1989). The plasmid vector may also include aselectable marker such as the β-lactamase gene for ampicillinresistance, provided that the marker polypeptide does not adverselyeffect the metabolism of the organism being treated. The cassette canalso be bound to a nucleic acid binding moiety in a synthetic deliverysystem, such as the system disclosed in WO 95/22618.

[0070] If desired, the preselected compound, e.g. a nucleic acid such asDNA may also be used with a microdelivery vehicle such as cationicliposomes and adenoviral vectors. For a review of the procedures forliposome preparation, targeting and delivery of contents, see Manninoand Gould-Fogerite, BioTechniques, 6, 682 (1988). See also, Felgner andHolm, Bethesda Res. Lab. Focus 11(2), 21 (1989) and Maurer, R. A.,Bethesda Res. Lab. Focus 11 (2), 25 (1989).

[0071] Replication-defective recombinant adenoviral vectors, can beproduced in accordance with known techniques. See, Quantin, et al.,Proc. Natl. Acad. Sci. USA 89, 2581-2584 (1992); Stratford-Perricadet,et al., J. Clin. Invest. 90, 626-630 (1992); and Rosenfeld, et al.,Cell, 68, 143-155 (1992).

[0072] The effective dose of the nucleic acid will be a function of theparticular expressed protein, the target tissue, the patient and his orher clinical condition. Effective amount of DNA are between about 1 and4000 μg, more preferably about 1000 and 2000, most preferably betweenabout 2000 and 4000.

[0073] Alternatively, the EC progenitors may be used to inhibitangiogenesis and/or neoplastic growth by delivering to the site ofangiogenesis a cytotoxic moiety coupled to the cell. The cytotoxicmoiety may be a cytotoxic drug or an enzymatically active toxin ofbacterial, fungal or plant origin, or an enzymatically activepolypeptide chain or fragment (“A chain”) of such a toxin. Enzymaticallyactive toxins and fragments thereof are preferred and are exemplified bydiphtheria toxin A fragment, non-binding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alphasarcin, certain Alcurites fordii proteins,certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII andPAP-S), Momordica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,and enomycin, Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAPare preferred.

[0074] Conjugates of the EC progenitors and such cytotoxic moieties maybe made using a variety of coupling agents. Examples of such reagentsare N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters such as dimethyladeipimidate HCI, active esters such as disuccinimidyl suberate,aldehydes such as glutaradehyde, bis-azido compounds such asbis(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene2,6-diisocyante, and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene.

[0075] The enzymatically active polypeptide of the toxins may berecombinantly produced. Recombinantly produced ricin toxin A chain(rRTA) may be produced in accordance with the methods disclosed in PCTW085/03508. Recombinantly produced diphtheria toxin A chain andnon-binding active fragments thereof are also described in PCTW085/03508.

[0076] The methods of the present invention may be used to treat bloodvessel injuries that result in denuding of the endothelial lining of thevessel wall. For example, primary angioplasty is becoming widely usedfor the treatment of acute myocardial infarction. In addition,endovascular stents are becoming widely used as an adjunct to balloonangioplasty. Stents are useful for rescuing a sub-optimal primary resultas well as for diminishing restenosis. To date, however, the liabilityof the endovascular prosthesis has been its susceptibility to thromboticocclusion in approximately 3% of patients with arteries 3.3 mm orlarger. If patients undergo stent deployment in arteries smaller thanthis the incidence of sub-acute thrombosis is even higher. Sub-acutethrombosis is currently prevented only by the aggressive use ofanticoagulation. The combination of vascular intervention and intenseanticoagulation creates significant risks with regard to peripheralvascular trauma at the time of the stent/angioplasty procedure.Acceleration of reendothelialization by adminstration of EC progenitorsto a patient undergoing, or subsequent to, angioplasty and/or stentdeployment can stabilize an unstable plaque and prevent re-occlusion.

[0077] The method of the present invention may be used in conjunctionwith the method for the treatment of vascular injury disclosed inPCT/US96/15813.

[0078] In addition, the methods of the present invention may be used toaccelerate the healing of graft tissue, e.g., vascular grafts.

[0079] In a further aspect, the methods of the invention may be used tostimulate tissue regeneration and in particular, cardiac regeneration.For example, autologous progenitor cells are provided to patients inneed of cardiac tissue regeneration (e.g., such as patients withcoronary artery disease (CAD), myocardial infarctions, etc.), andcontacted with one or more cardiomyocytes, thereby stimulating thedifferentiation of the progenitor cells to form additionalcardiomyocytes. Conversion of progenitor cells to cardiomyocytes can bemonitored by evaluating the expression of appropriate markers (e.g.,cardiac troponin I, atrial natriuretic peptide, α-sarcomeric actinin,MEF-2, and the like) and/or the uptake of diacylated LDL (sinceendothelial progenitor cells can take up diacylated LDL butcardiomyocytes cannot), measuring calcium transits, etc. Contacting mayoccur in vivo or ex vivo. Endothelial progenitor cells for stimulatingcardiac regeneration may be delivered to cardiac tissue by injection,preferably, in situ (e.g., via an endocardial delivery catheter or othersuitable medical access device).

[0080] Endothelial progenitors can be selected for any of theabove-described methods by obtaining mononuclear cells (e.g., from bloodor bone marrow), and selecting for one or more, two or more, or three ormore of the following markers: CD34⁺, Flk-1⁺, tie-2⁺′, CD31⁺, selectin,endothelial marker proteins (such as von Willebrand factor, vascularendothelial cadherin, and endothelial nitric oxide synthase), or one ormore of the following properties: uptake of diacetylated LDL(DiI-acLDL), and lectin binding.

[0081] In one preferred aspect, the markers: CD34⁺, Flk-1⁺, tie-2⁺′ areused.

[0082] The present invention also includes pharmaceutical products forall the uses contemplated in the methods described herein. For example,there is a pharmaceutical product, comprising nucleic acid encoding anendothelial cell mitogen and EC progenitors, in a physiologicallyacceptable administrable form.

[0083] The present invention further includes a kit for the in vivosystemic introduction of an EC progenitor and an endothelial cellmitogen or nucleic acid encoding the same into a patient. Such a kitincludes a carrier solution, nucleic acid or mitogen, and a means ofdelivery, e.g., a catheter or syringe. The kit may also includeinstructions for the administration of the preparation.

EXAMPLES

[0084] The present invention is further illustrated by the followingexamples. These examples are provided to aid in the understanding of theinvention and are not construed as a limitation thereof.

Example 1

[0085] Method and Materials

[0086] Human peripheral blood was obtained using a 20 gauge intravenouscatheter, discarding the first 3 ml. Leukocyte fraction of blood wasobtained by Ficoll density gradient centrifugation and plated on plastictissue culture for 1 hr to avoid contamination by differentiatedadhesive cells.

[0087] Fluorescent activated cell sorting (FACS) was carried out with>1×10⁶ CD34 positive and negative mononuclear blood cells (MB^(CD34+),MB^(CD34−)) Cells were analyzed with Becton-Dickinson FACS sorter andthe lysis II analysis program using antibodies to CD34 (Biodesign).

[0088] M-199 medium with 20% FBS and bovine brain extract (Clonetics)was used as standard medium for all cell culture experiments.

[0089] C57BL/6Jx129/SV background male mice (Hirlan), 3 mo old and 20-30g, were used in these experiments (n=24). Animals were anesthetized with160 mg/kg intraperitoneally of pentobarbital. The proximal end of onefemoral artery and distal portion of the corresponding saphenous arterywere ligated, following which the artery, as well as all side-branches,were dissected free and excised. (All protocols were approved by St.Elizabeth's Institutional Animal Care and Use Committee.)

[0090] New Zealand White rabbits (3.8-4.2 kg, n=4, Pine Acre Rabbitry)were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine(0.8 mg/kg) following premedication with xylazine (2 mg/kg). After alongitudinal incision, the femoral artery was dissected free along itsentire length; all branches of the femoral artery were also dissectedfree. After ligating the popliteal and saphenous arteries distally, theexternal iliac artery proximally and all femoral arterial branches, thefemoral artery was completely excised (Takeshita, et al, J. Clin.Invest. 93, 662 (1994); Pu, et al, Circulation 88, 208 (1993); Baffour,et al, J. Vasc. Surg. 16, 181 (1992)).

[0091] Isolation and Analysis

[0092] CD34 positive mononuclear blood cells (MB^(CD34+)) were isolatedfrom peripheral blood by CD34 antibody-coated magnetic beads (Dynal) asdescribed above.

[0093] FACS analysis indicated that 15.9±3.3% of selected cells versus<0.1% of the remaining cells expressed CD34. Depleted (MB^(CD34+)) cellswere used as controls. Flk-1 antibody was used for magnetic beadselection of Flk-1 positive mononuclear blood cells (MB^(Flk1+)).

[0094] MB^(CD34+) and MB^(CD34−) were plated separately in standardmedium on tissue culture plastic, collagen type I, or fibronectin. Whenplated on tissue culture plastic or collagen at a density of 1×10³/mm²,a limited number of MB^(CD34+) attached, and became spindle shaped andproliferated for 4 wks. A subset of MB^(CD34+) plated on fibronectinpromptly attached and became spindle shaped within 3 days (FIG. 1A); thenumber of attaching cells (AT^(CD34+)) in culture increased with time(FIG. 2). Attached cells were observed only sporadically among culturesof MB^(CD34−), including cells followed for up to 4 wks onfibronectin-coated plates.

[0095] To confirm that spindle-shaped cells were derived from CD34positive cells, MB^(CD34+) were labeled with the fluorescent dye, DiI,and co-plated with unlabeled MB^(CD34−) on fibronectin at an overalldensity of 5×10³/mm²; ratio of the two cell types was identical to thatof the original mononuclear cell population (1% MB^(CD34+), 99%MB^(CD34−)). Seven days later, DiI-labeled cells derived fromMB^(CD34+), initially accounting for only 1% of blood cells, accountedfor 60.3±4.7% of total attaching cells analyzed by FACS. Co-incubationwith MB^(CD34−) increased proliferation to >10× MB^(CD34+) plated aloneat a cell density of 5×10/mm² cell (d 3=131.3±26.8 vs 9.7±3.5/mm²).MB^(CD34+)/MB^(CD34−) co-cultures also enhanced MB^(CD34+)differentiation, including formation of cellular networks and tube-likestructures on fibronectin-coated plates (FIGS. 1B, C). These structuresconsisted principally of DiI-labeled MB^(CD34+) derived cells (FIG. 1C).Moreover, within 12 h of co-culture, multiple cluster formations wereobserved (FIG. 1D), consisting principally of DiI-labeled MB^(CD34+)derived cells (FIG. 1E). These clusters were comprised of round cellscentrally, and sprouts of spindle-shaped cells at the periphery. Theappearance and organization of these clusters resembled that of bloodisland-like cell clusters observed in dissociated quail epiblastculture, which induced ECs and gave rise to vascular structures in vitro(Flamme and Risau, 1982, supra). AT^(CD34+) at the cluster peripherywere shown to take up DiI-labeled acetylated LDL, characteristic of EClineage (Voyta, et al., J. Cell Biol. 99, 2034 (1984)), whereas theround cells comprising the center of cluster did not (FIGS. 1F,G); thelatter detached from the cluster several days later. Similar findingswere observed in the experiments using MB^(Flk1+).

[0096] Expression of Leukocyte and EC Markers

[0097] To further evaluate progression of MB^(CD34+) to an EC-likephenotype, cells were assayed for expression of leukocyte and ECmarkers. Freshly isolated MB^(CD34+) versus AT^(CD34+) cultured atdensities of 1×10³ cell/mm² for 7 days were incubated withfluorescent-labeled antibodies and analyzed by FACS (FIG. 3). Leukocytecommon antigen, CD45, was identified on 94.1% of freshly isolated cells,but was essentially lost by 7d in culture (FIG. 3). Augmented expressionof UEA-1, CD34, CD31, Flk-1, Tie-2 and E-selectin—all denoting EClineage (Millauer, et al, Cell 72, 835 (1993); Yamaguchi, et al.,Development 118, 489 (1993); Miettinen, et al., Am. J. Clin. Pathol. 79,32 (1983); Jaffe, et al., J. Clin. Invest. 52, 2745 (1973); Newman, etal, Science 247, 1219 (1990); Vecchi, et al, Eur. J. Cell Biol. 63, 247(1994); TSato, et al, Nature 376, 70 (1995); Schnurch and Risau,Development 119, 957 (1993); Bevilacqua, Annu. Rev. Immuno. 11, 767(1993))—was detected among AT^(CD34+) after 7 days in culture, comparedto freshly isolated MB^(CD34+). CD68 expression, suggestingmonocyte/macrophage lineage, was limited to 6.0±2.4% cells.

[0098] Expression of Factor VIII, UEA-1, CD31, ecNOS, and E-selectin wasalso documented by immunohistochemistry for AT^(CD34+) after 7 daysculture (data not shown). After 3, 7, and 14 days in culture, more than80% AT^(CD34+) took up DiI-labeled acLDL (Voyta, 1984, supra).

[0099] ECs uniquely express endothelial constitutive nitric oxidesynthase (ecNOS). Accordingly, MB^(CD34+), MB^(CD34−) and AT^(CD34+)were investigated for expression of ecNOS by RT-PCR (Janssens, et al.,J. Biol. Chem. 267, 14519 (1992); Lamos, et al., Proc. Natl. Acad. Sci.USA 89, 6348 (1992)). ecNOS mRNA was not detectable among MB^(CD34−) andwas present at very low levels in freshly isolated MB^(CD34+)+(FIG. 4).In AT^(CD34+) cultured for 7d, however, ecNOS mRNA was markedlyincreased (FIG. 5). Functional evidence of ecNOS protein in AT^(CD34+)was documented by measurement of nitric oxide in response to theEC-dependent agonist, acetylcholine (Ach), and the EC-specific mitogen,vascular endothelial growth factor (VEGF) (FIG. 5); the latterparenthetically constitutes evidence for a functional Flk-1 receptor aswell among AT^(CD34+).

[0100] Cell-Cell Interaction

[0101] Cell-cell interaction is considered to play a decisive role incell signaling, differentiation, and proliferation during hematopoiesis(Torok-Storb, Blood 72, 373 (1988); N. Dainiak, Blood 78, 264 (1991))and angiogenesis (Folkman and Klagsbrun, Science 235, 442 (1987); Hynes,Cell 48, 549 (1987); Brooks, et al., Science 264, 569 (1994);Friedlander, et al, Science 270, 1500 (1995)). To study the impact ofMB^(CD34+) interaction with mature ECs on the differentiation ofMB^(CD34−) into an EC-like phenotype, DiI-labeled MB^(CD34+) were platedon a confluent HUVEC monolayer. Adherent, labeled cells were foundthroughout the culture within 12 h (FIG. 6A), and increased in numberfor up to 3d (FIG. 6B). When incubated with 50 ng/ml VEGF and 10 ng/mlbFGF, a meshwork of cord-like structures comprised of both DiI-labeledand unlabeled cells could be seen within 3 d after co-culture (FIG. 6C).Both cell types were then re-seeded on Matrigel (Becton Dickinson)coated slides and within 12 h demonstrated formation of capillarynetworks comprised of DiI-labeled MB^(CD34+) derived cells and HUVECs(FIG. 6D). To facilitate cell-cell interaction, HUVECs were pre-treatedwith TNF-α (Simmons, et al, Blood 80, 388 (1992); Liesveld, et al.,Leukemia 8, 2111 (1994)), resulting in increased numbers of AT^(CD34+)(FIG. 6E); synergistic augmentation was observed upon co-incubation withVEGF. Identically treated co-cultures of HUVECs and DiI-labeledMB^(CD34−) yielded desquamated labeled cells and/or no cords. Similarfindings were observed when EC precursors were isolated usingMB^(Flk1+).

[0102] In Vivo Angiogenesis

[0103] Previous studies have established that ECs constitute theprincipal cell responsible for in vivo angiogenesis (Folkman, et al.,1985, surpra). To determine if MB^(CD34+) can contribute to angiogenesisin vivo, we employed two previously characterized animal models ofhindlimb ischemia. For administration of human MB^(CD34+),C57BL/6Jx129/SV background athymic nude mice were employed to avoidpotential graft-versus host complications. Two days later, when the limbwas severely ischemic, mice were injected with 5×10⁵ DiI-labeled humanMB^(CD34+) or MB^(CD34−) via the tail vein. Histologic sections of limbsexamined 1, 2, 4, and 6 wks later for the presence of DiI labeled cellsrevealed numerous DiI-labeled cells in the neo-vascularized ischemichindlimb. Labeled cells were more numerous in MB^(CD34+) versusMB^(CD34−) injected mice, and almost all labeled cells appeared to beintegrated into capillary vessel walls (FIGS. 8A, C, E, G).

[0104] No labeled cells were observed in the uninjured limbs of eitherMB^(CD34+) or MB^(CD34−) injected mice. DiI labeled cells were alsoconsistently co-labeled with immunostains for UEA-1 lectin (FIG. 8B),CD31 (FIG. 8D), and Tie-2 (FIG. 8F). In contrast, in hindlimb sectionsfrom mice injected with MB^(CD34−), labeled cells were typically foundin stroma near capillaries, but did not form part of the vessel wall,and did not label with UEA-1 or anti-CD31 antibodies (FIGS. 8G,H).

[0105] A transgenic mouse overexpressing β-galactosidase was then usedto test the hypothesis that homologous grafts of EC progenitors couldcontribute to neovascularization in vivo. Flk-1 cell isolation was usedfor selection of EC progenitors due to lack of a suitable anti-mouseCD34 antibody. Approximately 1×10⁴ MB^(Flk1+) were isolated from wholeblood of 10 β-galactosidase transgenic mice with B6,129 geneticbackground. MB^(Flk1+) or the same number of MB^(Flk1−) were injectedinto B6,129 mice with hindlimb ischemia of 2 days duration.Immunostaining of ischemic tissue for β-galactosidase, harvested 4 wksafter injection, demonstrated incorporation of cells expressingβ-galactosidase in capillaries and small arteries (FIG. 8I); these cellswere identified as ECs by staining with anti-CD31 antibody and BS-1lectin.

[0106] Finally, in vivo incorporation of autologous MB^(CD34+) into fociof neovascularization was tested in a rabbit model of unilateralhindlimb ischemia. MB^(CD34+) were isolated from 20 ml of blood obtainedby direct venipuncture of normal New Zealand white rabbits immediatelyprior to surgical induction of unilateral hindlimb ischemia. Immediatelyfollowing completion of the operative procedure, freshly isolatedautologous DiI-labeled MB^(CD34+) were re-injected into the ear vein ofthe same rabbit from which the blood had been initially obtained. Fourwks after ischemia, histologic sections of the ischemic limbs wereexamined. DiI-labeled cells were localized exclusively to neovascularzones of the ischemic limb, incorporated into capillaries andconsistently expressing CD31 and UEA-1 (FIGS. 8J,K).

[0107] Consistent with the notion that HSCs and ECs are derived from acommon precursor, our findings suggest that under appropriateconditions, a subpopulation of MB^(CD34+) or MB^(Flk−1+) candifferentiate into ECs in vitro. Moreover, the in vivo results suggestthat circulating MB^(CD34+) or MB^(Flk1+) in the peripheral blood mayconstitute a contingency source of ECs for angiogenesis. Incorporationof in situ differentiating EC progenitors into the neovasculature ofthese adult species is consistent with vasculogenesis, a paradigmotherwise restricted to embryogenesis (Risau, et al, Development 102,471 (1988); Pardanaud, et al., Development 105, 473 (1989); Flamme, W.Risau, 1992, supra). The fact that these cells do not incorporate intomature blood vessels not undergoing angiogenesis suggests that injury,ischemia, and/or active angiogenesis are required to induce in situdifferentiation of MB^(CD34+) to ECs.

Example II

[0108] EC Progenitors Augment Reendothelialization

[0109] Following balloon injury, a denuded rat carotid artery wasimmediately excised and placed in culture in HUVEC medium, and DiIlabeled CD34+ EC progenitor cells were seeded onto the artery. After 1wk, the artery was washed with PBS to remove non-adherent cells.Consistent with the ability of CD34+ cells to differentiate intofiltrating cells, DiI labeled cells were found within the smooth musclecell layer of the artery. Scanning electron microscopy of the intimalsurface, however, showed that DiI-labeled cells also had adhered to thedenuded arterial surface, assuming a morphology suggestive of ECs (FIG.9). DiI labeled cells also incorporated into the capillary-like sproutsat the bare ends of the excised arterial segment, suggesting that CD34+cells may be capable of participating in angiogenesis as well.

[0110] To determine if exogenously administered CD34+ EC progenitorcells can contribute to reendothelializationr of a denuded arterialsurface in vivo, freshly isolated human CD34+ or CD34− cells were DiIlabeled and seeded onto a denuded carotid artery of a nude rat.Following balloon denudation, 1.0×10⁶ labeled cells in PBS wasintroduced into the denuded artery via a 22G catheter, which remained inthe artery for 30 min before the needle was withdrawn. The externalcarotid artery was then ligated, the common and internal carotidarterial ligatures removed, and the incision closed. The next day therat was anesthetized and the vasculature perfusion fixed with HistoChoice (Amresco). The denuded arterial segment was excised and examinedfor the presence of adherent DiI labeled cells, which were identified inarteries seeded with CD34+ cells, but not CD34− cells.

[0111] The f references, patents, patent applications, and internationalapplications cited herein are incorporated herein by reference in theirentireties.

[0112] This invention has been described in detail including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of this disclosure, maymake modifications and improvements thereon without departing from thespirit and scope of the invention as set forth in the claims.

What is claimed is:
 1. A method for reducing undesired angiogenesiscomprising administering an endothelial progenitor cell which is CD34⁺,Flk-1⁺, and tie-2⁺ and a compound that inhibits angiogenesis, to alocation in need of reduced angiogenesis.
 2. A method for isolating anendothelial progenitor cell which is CD34⁺, Flk-1⁺, and tie-2⁺comprising: (a) obtaining human mononuclear cells; (b) selecting cellsthat express two or more of the following markers: CD34, Flk-1, tie-2′,CD31, UEA-1, selectin, von Willebrand factor, vascular endothelialcadherin, endothelial nitric oxide synthase and or one or more of thefollowing properties: uptake of diacetylated LDL (DiI-acLDL), and lectinbinding and (c) isolating the cells.
 3. A pharmaceutical compositioncomprising an endothelial cell (EC) progenitor which comprises or iscoupled to a cytotoxic agent, and a pharmaceutical carrier, wherein theEC progenitor is CD34⁺, Flk-1⁺, and tie-2⁺.
 4. A pharmaceuticalcomposition comprising an endothelial cell (EC) progenitor whichcomprises or is coupled to a co-stimulatory molecule for stimulating animmune reaction, and a pharmaceutical carrier, wherein the EC progenitoris CD34⁺, Flk-1⁺, and tie-2⁺.
 5. A pharmaceutical composition comprisingan endothelial cell (EC) progenitor which comprises or is coupled to anantitumor drug, and a pharmaceutical carrier wherein the EC progenitoris CD34⁺, Flk-1⁺, and tie-2⁺.
 6. A pharmaceutical composition comprisingan endothelial cell (EC) progenitor which comprises or is coupled to anangiogeneisis inhibitor, and a pharmaceutical carrier wherein the ECprogenitor is CD34⁺, Flk-1⁺, and tie-2⁺.
 7. A pharmaceutical compositioncomprising an endothelial cell (EC) progenitor, an endothelial cellmitogen, and a pharmaceutical carrier wherein the EC progenitor isCD34⁺, Flk-1⁺, and tie-2⁺.
 8. A method for reducing peripheral vasculartrauma during a stent or angioplasty procedure, comprising performing astent or angioplasty procedure on a patient and administering a modifiedendothelial progenitor cell which is CD34⁺, Flk-1⁺, and tie-2⁺ to thepatient before or after the procedure.
 9. A method for delivering anagent to a site of angiogenesis in a patient comprising: administeringto the patient an endothelial cell (EC) progenitor cell which comprisesor is coupled to the agent, wherein the EC progenitor is CD34⁺, Flk-1⁺,and tie-2⁺.
 10. A method of screening for the presence of ischemictissue or vascular injury in a patient comprising contacting the patientwith a labelled endothelial cell (EC) progenitor cell which is CD34⁺,Flk-1⁺, and tie-2⁺ and detecting the labelled cells at the site of theischemic tissue or vascular injury.
 11. A pharmaceutical compositioncomprising an endothelial cell (EC) progenitor which is CD34⁺ andcapable of differentiating into a CD34⁺, Flk-1⁺, and tie-2⁺ cell and oneor more agents for stimulating differentiation of the CD34⁺ endothelialcell (EC) progenitor into a CD34⁺, Flk-1⁺, and tie-2⁺ endothelial cell(EC) progenitor cell.
 12. A pharmaceutical composition comprising anendothelial cell (EC) progenitor which is CD34⁺ and capable of taking upacetylated LDL Flk-1⁺ and binding to fibronectin.
 13. A method fordelivering an agent to a site of angiogenesis in a patient comprising:administering to the patient, an endothelial cell (EC) progenitor cellwherein the EC progenitor is CD34⁺ and capable of differentiating into aCD34⁺, Flk-1⁺, and tie-2⁺cell, providing one or more agents forstimulating the differentiating.
 14. A method for improving orpreserving left ventricular (LV) function comprising injecting anendothelial cell (EC) progenitor cell which is CD34⁺ into the leftventricle of a heart.
 15. A method for improving or preserving cardiacfunction comprising identifying an area of hibernating cardiac tissue ina heart and contacting the tissue with at least one endothelial cell(EC) progenitor cell which is CD34⁺.
 16. A method for inducing orenhancing regeneration of cardiac tissue comprising administering acomposition comprising endothelial progenitor cells which are CD34⁺ to aheart in need of cardiac regeneration.
 17. A method for enhancing orstimulating blood vessel formation and/or cellular regeneration in aheart comprising delivering an endothelial progenitor cell which isCD34⁺ to heart tissue in need of blood vessel formation and/or cellularregeneration by introduction of a catheter percutaneously to theendocardial surface of the heart in proximity to the tissue in need. 18.A method for expanding a population of endothelial progenitor cellscomprising: a. providing a population of cells which comprises at leastone endothelial cell; b. contacting the population with one or morepurified endothelial progenitor cells which are CD34⁺ and capable ofdifferentiating into CD34⁺, Flk-1⁺, and tie-2⁺ cells; c. culturing thepopulation in the presence of a mitogen; thereby expanding cells whichare CD34⁺, Flk-1⁺, and tie-2⁺.